Study On The Effect Of Formonetin On Cancer Of Colon And Its Mechanism

Colorectal cancer(CRC)is one of the most common diseases of the digestive system in the world.It includes colon cancer and rectal cancer,which are also collectively called carcinoma of large intestine in the clinic.Colon cancer cells and rectal cancer cells have similar biological characteristics,so the clinical treatment principles are basically same.The incidence of Colorectal cancer ranks third in malignant tumors,which lists after lung cancer and breast cancer,and its mortality is ranked fourth in most common malignant tumor in the world.In China,the incidence and mortality of colorectal cancer are among the top five.In 2015,the incidence of colorectal cancer in China was approximately 37.6 per 100,000 and the mortality was approximately 19.1 per 100,000.And,as the age increases,the risk of colorectal cancer gradually increases.In recent years,with the arrival of the changes in lifestyle,eating habits,and the aging of the population,the prevalence and mortality of colorectal cancer in China has gradually increased.Active treatment of colorectal cancer can significantly increase their 5-year survival rate.Therefore,it is particularly important to conduct in-depth studies on the treatment of colorectal cancer.The main current treatment for colorectal cancer are surgery and chemotherapy.After radical resection of colorectal cancer,50%of patients relapse or metastasis and die.Although chemotherapeutic drugs can improve the clinical efficacy of colorectal cancer,chemotherapeutic drugs not only act on tumor cells,but also affect normal human cells,which destroy rapidly dividing tumor cells and simultaneously destroys hair follicle cells,oral and gastrointestinal mucosal cells,red blood cells,and white blood cells,etc.Therefore,the non-specific action of chemotherapeutic drugs can cause many side effects.More and more research shows that traditional Chinese medicine has positive significance for the treatment of tumors and has good tolerability.Therefore,it is particularly important to actively develop Chinese medicine preparations with antitumorigenic effects and small side effects.Formononetin(Form)which is a bioactive isoflavone that inhibits the proliferation of multiple cancer cells and induces them apoptosis.However,There are no enough and in-depth research reports yet.In this research,SW1116 and HCT116 cell lines of colon cancer were selected as the research objects to study the inhibitory effect of formononetin on the growth proliferation and invasive ability of cancer of colon cells,and to analyze its possible mechanism.On this basis,the effect of formononetin on animal cancer of colon in vivo was studied using a transplanted tumor animal model,which provides experimental evidence for the treatment of colorectal cancer with formononetin.Part one Study on the effect of Formonetin on the growth,proliferation and invasion of cancer of colon cell.Objective:The effects of Formonetin on proliferation,invasion and cell cycle of SW1116 and HCT116 were detected,so to make it clear that the effect of Form on Cancer of colon.Methods:SW1116 and HCT116 cell lines were cultured regularly.MTT was performed to determine the effect of Form on the proliferation of the involved cell lines,in addition to that,Transwell assay and flow cytometry were adopted for the detections of cell invasion and cell cycle treated with form.Results:1.The inhibition effect of Form on the growth of cancer of colon cellsForm was observed to inhibit the growth of SW1116 and HCT116 in manners of dose and time effect,namely,the inhibition effect was increasing as the dose arose and the time went by,and the difference was statistic significant(P<0.05)by means of ANOV.Form could also inhibit the growth of NCM460 cell,but the inhibition rate to NCM460(18.26±3.09%)was much weaker than those to SW1116 and HCT116 at 200?M Form treated for 72h(83.67±4.32%,86.2±5.48%).2.Effect of Form on cell cycle and proliferation index of cancer of colon cellsSW1116 and HCT116 cells were cultured,and treated with various concentrations of Form(20,50,100?M)for 48h,FCM was operated for the cell Cyclin analysis.The results indicated that the proportion of G0/G1 stage cell was increasing as the concentration of Form increasment.The ratio of G0/G1in SW1116 and HCT116 with Form treatment for 48h were 79.2±4.8%and79.7±5.7%respectively.It could be concluded that Form arrest SW1116 and HCT116 at G0/G1 stage.The PI of SW1116 and HCT116 was observed to be in a persistent decreasment as the increasment of Form.3.The Effect of Form on the invasion of cancer of colon cellsNCM460 and SW1116,HCT116 cells were cultured and treated by various Form concentrations(50,100?M)for 24h,and transwell assay was performed for the detection of cell invasion.The results demonstrated that Form could inhibit the invasion of SW1116 and HCT116 cells at a dose-effect manner,while Form had no effect on the invasion of NCM460 cell.Summary:1.Form could inhibit the growth of cancer of colon cells at dose and time effect manners.2.Form could induce cancer of colon cell arrest at G0/G1 stage,and inhibit the proliferation of cancer of colon cells at a dose-effect manner.3.Form could inhibit the invasion of cancer of colon cells at a dose effect manner.Part two Study on the mechanism of Formonetin on cancer of colon cells.Objective:By detecting the expressions of miR-149,EphB3m RNA,signal moleculars,cell cycle and invasion related proteins in cancer of colon cells treated by Form,the possible mechanism of tumor inhibition by Form was discovered.Methods:Western Blot was performed for the detection of the expression of CyclinD1,MMP-2/9 and signal moleculars as p-ERK,p-AKT,p-PI3K and p-STAT3.The effects of Form on the expressions of miR-149 and EphB3 mRNA were analyzed by means of RT-PCR.The roles of miR-149 and EphB3 mRNA gene to the biological behavior of SW1116 and HCT116treated by Form were demonstrated by transient transfection.Results:1.The effects of Form on the expressions of CyclinD1,Cyclin B1,MMP-2/9 and AKT/PI3K/ERK/STAT3 in SW1116 and HCT116 cellsForm could inhibit the expression of CyclineD1 of cancer of colon cells at a dose-effect manner,but had no effect on CyclinB1.Form was observed to down-regulated the expressions of MMP-2 and MMP-9 at a dose-effect manner.Form could down-regulate the expressions of p-AKT,p-PI3K and p-STAT3,but had no effect on p-ERK,and there were no statistics differences about the total ERK,AKT,PI3K and STAT3 in SW1116 and HCT116 cells between before and after treatment with Form.2.The effects of Form on the expressions of miR-149 and EphB3 mRNA in NCM460,SW1116 and HCT116 cellsCompared to control,Form could up-regulate the expression of miR-149in SW1116 and HCT116 cells at a dose-effect manner(P<0.05).But it demonstrated an adversary result about the effect of Form on EphB3 mRNA.Form had no effect on the expressions of miR-149 and EphB3 mRNA in NCM460 cell at lower concentration,while it could up-regulate the expression of miR-149 and down-regulate EphB3 at high concentration3.Verification of SW1116 and HCT116 cells transfected by miR-149 and EphB3 siRNACompared to control,the expressions of miR-149 in transfected SW1116and HCT116 cells were higher significantly,and it indicated that miR-149 was successfully transfected.The result of RT-PCR demonstrated that the expressions of EphB3 mRNA in siEphB3 200nM and siEphB3 300nM groups were higher than those of siEphB3 100nM and siRNA control,and there was no statistics difference between siEphB3 200nM group and siEphB3 300nM group.It could be concluded that 200nM siEphB3 induce silence of EphB3mRNA effectively.4.The effect of miR-149 mRNA transfection on the expression of EphB3in SW1116 and HCT116 cellsThe expressions of EphB3 in SW1116 and HCT116 cells transfected with miR-149 decreased significantly,and it indicated that miR-149 could inhibit the expression of EphB3.5.The effects of Form on the viability and invasion of SW1116 and HCT116 transfected with miR-149The viabilities of control,Form group,mimic-NC+Form group and mimic miR-149+Form group SW1116 cells were 1.0±0.0,0.39±0.14,0.41±0.18 and 0.18±0.06 respectively,the numbers of invasion cells were168.2±16.3,67.5±12.0,72.0±18.6 and 28.3±2.8.The viabilities of control,Form group,mimic-NC+Form group and mimic miR-149+Form group HCT116 cells were 1.0±0.0,0.41±0.16,0.42±0.28 and 0.19±0.07.The numbers of invasion cells were 146.8±11.5,56.2±5.6,58.8±7.2 and 26.7±5.1.The results indicated that Form inhibit the viability and invasion of SW1116and HCT116 transfected with miR-149.6.The effect of Form on the viability and invasion in SW1116 and HCT116 transfected with siEphB3The viabilities of control,Form,siNS+Form,siEphB3+Form group SW1116 cells were 1.0±0.0,0.49±0.17,0.51±0.24 and 0.11±0.02 respectively,and the numbers of invasion cells were 189.6±10.2,63.7±5.9,61.8±4.6 and11.2±1.8 respectively.The viabilities of control,Form,siNS_Form,siEphB3+Form group HCT116 were 1.0±0.0,0.52±0.19,0.51±0.10 and0.22±0.08 respectively,and the numbers of invasion cells were 171.5±12.0,72.3±9.5,69.4±8.2 and 12.2±5.0.The results indicated that Form inhibit the viability and invasion of SW1116 and HCT116 cells transfected with siEphB3.Summary:1.The inhibition of CyclinD1 contributed to cancer of colon cells arrest by Form,while the down-regulation of MMP-2 and MMP-9 might contribute to the decreament of cell invasion.Form could down-regulate the expressions of p-AKT,p-PI3K and p-STAT3 in cancer of colon cells.2.Form was observed to up-regulate the expression of miR-149 in cancer of colon cells,while down-regulate that of EphB3 mRNA.3.miR-149 mimic was successfully transfected into cancer of colon cells,and 200nM siEphB3 was effective silence EphB3 mRNA.4.The EphB3 was inhibited by miR-149 in cancer of colon cells.5.The viability and invasion of SW1116 and HCT116 cells were inhibited by Form after transfected with miR-149.6.The viability and invasion of SW1116 and HCT116 cells were inhibited by Form after silence EphB3 mRNA.Part three:Study on the effect of Formonetin on the growth of xenograft cancer of colon in nude miceObjective:The inhibition effect of Form on cancer of colon was studied with the help of xenograft in nude mice,and the possible mechanism of Form on tumor was reclarified.Methods:Xenograft cancer of colon in nude mice was established.The effect of Form on the volume and wet weight of Xenograft cancer of colon treated by Form were measured,and the tumor inhibition rate was caculated.Other than that,the pathological change of Xenograft cancer of colon was observed by HE stain,and positive PCNA cell was numerated,in addition,immunohistochemistry was performed for the observation of CyclinD1 and MMP-9 in Xenograft cancer of colon.Results:1.Effectiveness of adenovirus infected HCT116 cellRT-PCR was performed to verify the effectiveness of Ad-EphB3 and Ad-GFP infected HCT116 cell.Adenovirus successfully infected HCT116 cell in accordance with the evidence that the expression of EphB3 mRNA in cell infected by Ad-EphB3 was higher than that by Ad-GFP.2.Establishment of xenograft colon cancer in nude miceAbout 5 days after HCT116 and HCT116 infected by Ad-EphB3 were planted into left armpits of nude mice,local nodule could be touched,and the tumor volume was increasing in all of the animal models.The xenograft tumor was regular in shape and atypical leaflet.The surface of tumor was smooth at early stage,but they became dark as the enlargement of tumor,then necrosis and hemorrhage were observed.3.The change of body weight and physical conditionAll of the body weight of mice in the 3 groups increased during experiment period.The additional weight of mice in control,Ad-EphB3+Form and Form group were 4.82±0.62g,5.13±0.81 and 4.91±0.53g respectively.There were no statistic differences among the above 3 groups(P>0.05).The dietary was stable during experiment period,so were the mental state and movement.4.The inhibition effect of Form on the growth of xenograft tumor in nude mice4.1.The growth curve of xenograft t tumor in nude miceThe growth curve was drawn with treatment time as X-axial and tumor volume as Y-axial.The volumes of tumor in control were 60.05±9.2mm~3,102.2±10.2mm~3,207.2±19.8mm~3,265.0±24.3mm~3,421.2±40.7mm~3 and 640.0±45.1mm~3 at 11,14,17,20,23 and 26 days after treatment respectively.And those in Ad-EphB3+Form were 72.8±6.3mm~3,107.9±9.8mm~3,149.9±13.5mm~3,192.2±17.4mm~3,253.8±20.6mm~3 and 409.7±32.4mm~3 respectively,while they were 52.6±4.3mm~3,97.3±8.8mm~3,130.1±11.5mm~3,173.5±15.4mm~3,213.9±19.1mm~3 and 308.8±29.7mm~3 respectively in Form group.Both of the volumes of tumor in Form and Ad-EphB3+Form group were less than that in control,and the volume of tumor in Form group was less than that in Ad-EphB3+Form group,the differences were statistical significant(P<0.05).4.2.The effect of Form on the wet weight and inhibition rate of xenograft colon cancer in nude miceTumor was separated after the mice were killed 26 days after treatment,and the wet weights of tumor were measured.The wet weights of tumor of control,Ad-EphB3+Form and Form group were 0.78±0.10g,0.54±0.06 g and0.42±0.05 g respectively.Compared to control,the wet weights of tumor in Form and Ad-EphB3+Form group were lighter by ANOVA,and the wet weight of Form group was lighter than that in Ad-EphB3+Form group,and the differences were statistical significant(P<0.05).The inhibition rate of Form and Ad-EphB3+Form were 46.41±5.90%and30.26±7.88%respectively,and the difference was statistical significant by independent sample t-test.5.Morphology of xenograft colon cancer in nude mice treated with Form by light microscopeHE was operated for the observation of xenograft colon cancer.Observed by light microscope,the transplanted tumor cells showed obvious heterotypic changes,which existed differences in size of cells and the size,shape and staining of the nuclei.In addition,the mitoses were easy to see,and necrosis was found in the tumor tissue.compared to control,the necrosis area of tumor tissue in Form and Ad-EphB3+Form group were wider,and more serious of fiber tissue and inflammation cells infiltration.The above morphological items in Fomr group were more serious than those in Ad-EphB3+Form group.6.PCNA immunohistochemistry in xenograft colon cancer in nude mice treated with FormThe result of immunohistochemistry demonstrated that the positive cell numbers of PCNA stain in tumor tissue of Form and Ad-EphB3+Form groups were decreased dramatically when compared to that of control,and the positive PCNA stain cells in Form group was less than that in Ad-EphB3+Form group.7.CyclinD1 and MMP-9 immunohistochemistry of xenograft colon cancer in nude mice treated with FormImmunohistochemistry was performed for the observation of CyclinD1and MMP-9 in tumor tissue treated with Form.Compared to control,the expressions of CyclinD1 and MMP-9 in Form and Ad-EphB3+Form groups were lower,and both of the expressions of CyclinD1 and MMP-9 in Form group were lower than those in Ad-EphB3+Form group.Summary:1.Adenovirus successfully infected HCT116 cell.2.Xenograft colon cancer in nude mice was successfully established with100%cases.3.Form could inhibit the growth of xenograft colon cancer in nude mice,and EphB3 mRNA was likely to weaken its inhibition effect.4.Form could induce the necrosis of xenograft colon cancer in nude mice,and EphB3 mRNA was likely to weaken its effect.5.Form was observed to decrease the expressions of PCNA,CyclinD1and MMP-9 of xenograft colon cancer in nude mice,and EphB3 was likely to weaken its effect.Conclusions:1.Form could up-regulate the expression of miR-149 in cancer of colon cells,following that the expression of the target gene of miR-149,namely EphB3 mRNA was decreased.2.The expressions of signal moleculars p-AKT,p-PI3Kand p-STAT3 were inhibited by Form,and this might contribute to the inhibition effect of Form on cancer of colon cells.3.Form could inhibit the expression of CyclinD1 in colon cancer cells,and induce cell arrest at G0/G1 stage,as a result,the cell proliferation was inhibited,and it effected at a dose-effect manner.Other than that,Form could inhibit the invasion of cancer of colon cells by decreasing the expressions of MMP-2/9,and it effected at a dose-effect manner.4.Form was observed to inhibit the growth of xenograft colon cancer in nude mice.